iv

ANTIBIOTIC PURIFICATION BY USING IMA ADSORBENTS

ABDUL RAHIM BIN MOHD YUSOFF

Thesis submitted to the Faculty of Chemical and Natural Resources Engineering in

Partial Fulfillment of the requirement for the
Degree of Bachelor Engineering in Chemical Engineering

Faculty of Chemical and Natural Resources Engineering

University Malaysia Pahang

APRIL 2009

I declare that this thesis entitle Antibiotic Purification by Using IMA Adsorbents is
the result of my own research except as cited in the references. The thesis has not
been accepted for any degree and not concurrently submitted in candidature of any
degree

Signature

Name

: ABDUL RAHIM BIN MOHD YUSOFF

Date

: 30 APRIL 2009

vi

Special Dedication of This Grateful Feeling to My

Beloved father and mother;

Mr. Mohd Yusoff bin Hussin and Mrs. Rabainah binti Hashim

Loving brothers and sisters;

Shahiful, Shahrizal, Asrol, Zaidi, Zurianie,
Azmir, Huda, Azizah and Shalehuddin.

Supportive friends
For Their Love, Supports and Best Wishes

vii

ACKNOWLEDGEMENT
Bismillahirahmanirahim and Alhamdulillah. First and foremost, I want to
express gratitude to my mother, Madam Rabainah Binti Hashim, my father Mr.
Mohd Yusoff Bin Hussin and the rest of my family for their unconditional support
and encouragement.

I would like to thank my supervisor, Miss Suriyati Binti Saleh for her
invaluable advice and contribution to this work. Her insights and high standards have
definitely helped to shape this work. It is pleasure to have an advisor being so joyful
in her work.

I would like to also take this opportunity to thank all lectures who involved
directly and indirectly in helping me to complete this research. For personnel at
FKKSA clean room especially in Bio-processing lab and Analytical lab, Mr Anuar,
Miss Hafiza, Mr. Razak and also FKM laboratory staffs, Mr Jamiluddin and Mr
Khairidz Azuwar for all guidance, trust, assistance and constructive ideas.

Thank also to my friends with same supervisor, Miss Fidelia, Miss Farhani and Miss
Munirah for their moral supports and assistants. Thank you very much and hope our
friendship will last until forever.

Thank to former and present colleagues at Universiti Malaysia Pahang for

making and enjoyable working environment and giving me ideas, opinions and
advices. Thank you again.

viii

ABSTRACT

The intensity to achieve highly efficient and economical separation

process can be seen in developing of various methods in the recent year. While in
purification of antibiotic there are many methods use such as using High
performance

liquid

chromatography

(HPLC)

and

Counter-current

chromatography (CCC). The purpose of this research is to develop immobilized

metal ion affinity zeolite by using solid state ion exchange method to investigate
the effect of pH, types of adsorbent using different metal into rifampicin
adsorption capacity. Adsorption of rifampicin using zeolite has a greatly potential
due to ability to scale up easily, and highly selective. It was found that the
highest adsorption capacity for rifampicin occur at pH 8 with Zr-HBeta as
adsorbent. H-beta zeolite give highest adsorption capacity because it has higher
diameter size, surface area and pore volume compare to Y zeolite. Increasing the
surface area and pore volume will give better chances of rifampicin to adsorb
into adsorbent. Meanwhile, pH 8 gives the highest adsorption capacity because it
is closer with the pKa2 value of rifampicin which is 7.9. While zirconium is the
only transition metal containing both acidic and basic surface sites. So this will
make it gives better adsorption capacity of rifampicin compare with ferum and
nickel. The adsorption isotherm data of rifampicin was well correlated by the
Langmuir model.

ix

ABSTRAK

Keinginan yang tinggi untuk mencapai proses pemisahan yang ekonomi

dan berkecekapan tinggi dapat dilihat melalui penghasilan pelbagai cara sejak
kebelakangan ini. Terdapat pelbagai cara dalam penyulingan antibiotic seperti
HPLC and CCC. Tujuan kajian ini adalah untuk menyediakan ion logam tarikan
dimasukkan ke dalam zeolite menggunakan kaedah penukar ion berkeadaan
pepejal untuk melihat kesan pH, jenis penjerap daripada jenis logam yang
berlainan terhadap kapasiti penjerapan rifampicin. Penjerapan rifampicin
menggunakan zeolite mempunyai potensi yang besar kerana mudah dioperasikan
pada skala yang lebih besar, beroperasi secara berterusan dan mempunyai
kememilihan yang tinggi. Kapasiti penjerapan tertinggi untuk rifampicin adalah
pada pH 8 dengan mengunakan penjerap logam zirkonium. Zeolite H-beta
memberikan kapasiti penjerapan tertinggi kerana ianya mempunyai saiz
diameter, luas permukaan and isipadu pori yang lebih besar berbanding dengan
zeolite Y. Pertambahan luas permukaan serta isipadu pori akan memberikan
peluang yang lebih kepada rifampicin untuk menyerap ke dalam penjerap.
Dalam pada itu, pH 8 memberikan kapasiti penjerapan tertinggi kerana ianya
dekat dengan nilai pKa2 bagi rifampicin iaitu 7.9. Sementara itu, hanya
zirkonium sahaja logam peralihan yang mengandungi sifat asid dan alkali bagi
kedua-dua belah permukaan. Ini menjadikan zirkonium memberikan kapasiti
penjerapan rifampicin lebih baik berbanding dengan ferum dan nikel. Data
penjerapan rifampicin menunjukkan ianya menghampiri model Langmuir.

TABLE OF CONTENTS

CHAPTER

TITLE

PAGE

TITLE PAGE

iv

DECLARATION

DEDICATION

vi

ACKNOWLEDGE

vii

ABSTRACT

viii

ABSTRAK

ix

TABLE OF CONTENTS

LIST OF TABLE

xiii

LIST OF FIGURES

xiv

LIST OF SYMBOLS

xvi

LIST OF ABBREVIATIONS

xvii

LIST OF APPENDICES

xix

INTRODUCTION
1.1 Background of Study

1.2 Problem Statement

1.3 Objectives of Study

1.4 Scopes of Study

LITERATURE REVIEW
2.1 Antibiotics

xi

2.1.1 Major principle and definition

2.1.2 Antibiotic resistance

11

2.1.3 Categories of antibiotic

14

2.1.4 Antibiotics and chemotherapeutic agents

16

2.2 Zeolite

17

2.2.1 Natural and synthesis zeolite

20

2.2.2 Characteristics of natural and synthesis zeolite

24

2.2.3 Uses of zeolite

28

2.3 Metal Ion Affinity Chromatography (IMAC) Adsorbents

29

2.3.1 Principle of IMAC

30

2.3.2 Flexibility of IMAC

31

2.3.3 IMAC Adsorbents

32

2.4 Adsorption

33

2.4.1 Introduction

33

2.4.2 Adsorbent

34

2.4.3 Adsorption process

37

2.4.4 Adsorption theory

39

2.4.5 Adsorption Theorem

40

2.4.5.1 Langmuir equation

40

2.4.5.2 Freundlich equation

42

3 METHODOLOGY
3.1 Material

45

3.1.1General Chemical and Material

45

3.1.2 Adsorbent

45

3.1.2.1 Adsorbent selection criteria

46

3.1.3 Material selection

47

3.1.3.1 Zirconium

47

3.1.3.2 Ferum

48

3.1.3.3 Nickel

48

3.1.4 Rifampicin

49

3.2 Preparation of Immobilized Metal Ion Affinity

50

3.3 Solution Preparation

50

3.3.1 Antibiotic solution

50

xii

3.3.2 Buffer preparation

51

3.4 Experimental Procedures

51

3.5 Adsorption Isotherm Analysis

51

4 RESULT AND DISCUSSIONS

4.1 Introduction

53

4.2 Effect of pH

53

4.3 Effect of Adsorbents

55

4.4 Effect of Various Metal Ions

57

4.5 Adsorption Isotherm

58

4.5.1 Effect of pH

58

4.5.2 Effect of adsorbents

59

4.5.3 Effect of various metal ions

61

CONCLUSION AND RECOMMENDATION

5.1 Conclusion

63

5.2 Recommendation

64

REFERENCES

66

APPENDICES

68

xiii

LIST OF TABLES

TABLE NO.

TITLE

PAGE

2.1

Mechanisms of preventing antibiotic resistance

14

2.2

Classification of antibiotic by their structures

15

2.3

ZSM-Type zeolite

18

2.4

Properties of zeolite (natural & synthetic)

27

2.5

Classification of common adsorbents

35

2.6

Classification of pore sizes

37

4.1

Effect of pH on Langmuir constant for rifampicin

59

4.2

Effect of adsorbent on Langmuir constant forriampicin

60

4.3

Effect of various immobilized metal ion affinity adsorbents

61

on Langmuir constant for rifampicin.

xiv

LIST OF FIGURES

FIGURE NO.

TITLE

PAGE

2.1

Chemical structure of rifampicin

2.2

Chemical structure of some important penicillins

2.3

Chemical structure of cephalosporins

2.4

The bacterial mechanisms of antibiotic resistance

13

2.5

Image of clinoptilolite zeolite

21

2.6

Beta polytypes A(tetragonal, P4122,), B(monoclinic C2/c,)

23

and C(P42/mmc)
2.7

Y Zeolite crystal structure

24

2.8

Example graph for Langmuir isotherm

42

2.9

Example graph for Freundlich isotherm

44

3.1

Structure of rifampicin

50

4.1

Effect of pH on the adsorption of rifampicin

54

4.2

Effect of adsorbents on the adsorption to the zirconium ion

55

4.3

Effect of adsorbents on the adsorption to the ferum ion

56

4.4

Effect of adsorbent on the adsorption to the nickel ion

56

4.5

Effect of various metals on the adsorption onto Hbeta zeolite

57

4.6

Effect of various metals on the adsorption onto Y zeolite

58

4.7

Effect of pH on adsorption isotherm of rifampicin

59

onto Zr-Heta zeolite

4.8

Effect of adsorbent on adsorption isotherm of rifampicin

60

xv

4.9

Effect of various metal ions on adsorption isotherm of rifampicin 61

xvi

LIST OF SYMBOLS

Al

Aluminium

Concentration mM

Fe

Ferum

Kd

Langmuir adsorption parameter

pH

Negative logarithmic molar concentration of hydrogen ion, -log[H+]

pKa

Acid dissociation constant

Freundlich constant

Na

Sodium

Ni

Nickel

Solute concentration in adsorbent mmol/g

qm

Langmuir isotherm parameter mmol/g

Si

Silica

Zr

Zirconium

xvii

LIST OF ABBREVIATIONS

CASMAC

Cascade-mode multi-affinity chromatography

CCC

Counter-current Chromatography

CEC

Cation Exchange Capacity

CNS

Central Nervous System

DNA

Deoxyribonucleic acid

EDTA

Ethylenediaminetetraacetic acid

FTIR

Fourer Transform Infrared

H3PO4

Phosphoric acid

HSCCC

High Speed Counter-current Chromatography

IDA

Iminodiacetic acid

IMA

Immobilized metal ion affinity

IMAC

Immobilized metal ion affinity chromatography

IUPAC

International Union of Pure and Applied Chemistry

FDA

Food and Drug Administration

HPLC

High Performance Liquid Chromatography

K2CO3

Potassium carbonate

K2HPO4

Potassium Hydrogen Phosphate

KH2PO4

Potassium Dihydrogen Phosphate

KHCO3

Potassium hydrogen carbonate

LEC

Ligand Exchange Chromatography

NTA

Nitrilotriacetic acid

xviii

RNA

Ribonucleic acid

UV-VIS

Ultra Violet Visible

xix

LIST OF APPENDICES

APPENDIX

TITLE

PAGE

A.1

Preparation of antibiotic solution

68

A.2

Preparation of buffer Solution

69

A.3

Calibration curve for rifampicin initial adsorbance

70

CHAPTER 1

INTRODUCTION

1.1 Background of Study

Antibiotics are organic substances produced by special microorganisms or

other living systems. Generally, antibiotic are produced on an industrial scale using a
fermentation process and capable at low concentration of inhibiting the growth of, or
destroying another microorganism. Antibiotics have been isolated from numerous
sources

but

mainly

from

bacteria

(tetracyclines,

bacitracin,

polymyxin,

chloramphenicol, and streptomycin) and fungi (cephalosporins, penicillins).

Penicillin was the first antibiotic discovered by Sir Alexander Flemming in 1928. It
is derive from the Penicillium mold and acts by destroying the cell wall of bacteria.
The name penicillium was taken from the Latin penicillum meaning a painter's brush
because the fronds of the fungus were thought to look like a painter's brush.

Antibiotics are the most important bioactive and chemotherapeutic

compounds made by microbiological synthesis. They also include antimicrobial
compounds present in higher plants and animals. They have proven their significance
in varied fields like medicinal chemistry, agriculture and food industry.

Up to now about 40 000 antibiotics have been found and about 80 of them are
in therapeutic use. They are isolated primarily from metabolic products of living
cells. Various penicillins, cephalosporins and several other antibiotics are semisynthetic ones, which mean one part of the molecule, i.e. 6-amino penicillanic acid is

prepared from say penicillin G or penicillin V, followed by synthetic introduction of

an appropriate side chain.

Zeolites are crystalline, hydrated aluminosilicates of alkali and alkaline earth

cations characterized by an ability to hydrate/dehydrate reversibly and to exchange
some of their constituent cations with dissolved cations in solution, both without a
major change in structure. The ion exchange property of these minerals has
generated worldwide interest for use in diverse applications such as the treatment of
nuclear, municipal, and industrial waste water. Although commercial applications of
ion exchange processes have used mainly synthetic zeolites, the earliest studies of
ion exchange phenomena were based on observations of natural materials, including
natural zeolites.

Commercial adsorbents that display ultra porosity include activated carbons,

activated clays, inorganic gels, such as silica gel and activated alumina, and the
crystalline aluminosilicate zeolite. Activated carbons, activated alumina, and silica
gel do not possess an ordered crystal structure and consequently their pores are nonuniform. The distribution of the pore diameters within the adsorbent particles may
vary widely from 20 to several thousand Angstroms, as is the case for some activated
carbons. Hence, all molecular species, with the possible exception of high molecular
weight polymers, may enter the pores. Zeolite molecular sieves, on the other hand,
have pores of uniform size (310) which are uniquely determined by the unit
structure of the crystal. These pores will completely exclude molecules that are
larger than their diameter. J. W. McBain (1932) originated the term molecular
sieves to define porous solid materials that exhibit the property of acting as sieves
on a molecular scale.

Synthetic adsorbents are widely used as polymeric media for recovery and
separation of antibiotic or their intermediates, foods, etc. For example, they are used
for separation of antibiotics such as penicillin, cephalosporin and their derivatives,
because of their high adsorption capacity, mechanical strength and chemical stability
suitable for industrial operations.

Column operations are commonly adopted for those applications. In this

sense, the synthetic adsorbents are used as chromatographic separation media.
Therefore, both pore and chemical characteristics of the synthetic adsorbents will
affect the separation and adsorption capacity of target compounds.

1.2 Problem Statement

The development of an antibiotic is a long and costly proposal. It begins with

basic research designed to identify organisms, which produce antibiotic compounds.
During this phase, thousands of species are screened for any sign of antibacterial
action. When one is found, the species is tested against a variety of known infectious
bacteria. This is a complex procedure because thousands of antibiotic materials have
already been discovered. Repeatedly, scientists find that their new antibiotics are not
unique. If the material passes this phase, further testing can be done. This typically
involves clinical testing to prove that the antibiotic works in animals and humans and
is not harmful. If these tests are passed, the government agencies like the Food and
Drug Administration (FDA) must then approve the antibiotic as a new drug. This
whole process can take many years.

Normally production of an antibiotic depends on a fermentation process.

During fermentation, amounts of the antibiotic-producing organism are grown and
the organisms produce the antibiotic material, which can then be isolated for use as a
drug. Development of antibiotics necessitates isolation and purification of a desired
compound from a complicated matrix such as fermentation broth and crude extract.
Analysis of antibiotics in formulated and unformulated samples demand a highly
specific and rapid method as many antibiotics (e.g. -lactams) also have serious
stability problems.

There are many methods in separation of antibiotic. HPLC technology using

sophisticated equipments and refined adsorbents highly facilitate the isolation of
antibiotics; there are some drawbacks due to various complications arising from the

use of a solid support. Other method is Counter-current chromatography (CCC) is a

unique form of liquid partition chromatography which utilizes a separation column
free of solid support matrix. Because of this support-free system, the method
provides an important advantage over other chromatographic methods by eliminating
various complications including an adsorptive loss and deactivation of samples,
contamination, etc.

Immobilized metal ion affinity chromatography (IMAC) is one of the most

powerful separation methods available for protein fractionation. For antibiotic
separation, this method is use wisely yet. Other thing is traditional stationary phase
for IMAC are based on soft gel. But for this research, we will use some inorganic
material adsorbent.

1.3 Objective of Study

The purpose of this research is to use zeolite (H-beta, Y) as an immobilized
metal ion affinity stationary phase by using three different metal (zirconium, ferum
and nickel) and rifampicin as an antibiotic solution for antibiotic separation.

1.4 Scopes of Study

In order to achieve objectives, the scopes for this research are:

1. To study the effect of different metal use in IMA
2. To study type of zeolite
3. To study the effect of pH
4. To study the effect of antibiotic concentration

CHAPTER 2

LITERATURE REVIEW

2.1 Antibiotics

Antibiotics are chemical compounds used to kill or inhibit the growth of

infectious organisms. The antibiotic terms originally referred only to the organic
compounds that produced by bacteria or molds that are toxic to other
microorganisms. The term is now used freely to include synthetic and semi synthetic
organic compounds. Antibiotic refers generally to antibacterial, however, because
the term is loosely defined, it is preferable to specify compounds as being anti
malarial, anti viral, or anti protozoas. All antibiotics share the property of selective
toxicity: They are more toxic to an invading organism than they are to an animal or
human host. Penicillin is the most well-known antibiotic and has been used to fight
many infectious diseases, including syphilis, gonorrhea, tetanus, and scarlet fever.
Another antibiotic, streptomycin, has been used to combat tuberculosis.

Rifampicin (Figure 2.1) is the most important compound of rifamycin group

inhibits the growth of most gram-positive and some -negative microorganisms by
inhibiting their RNA synthesis. Rifampicin is a bactericidal antibiotic drug of the
rifamycin group. It is a semi synthetic compound derived from Amycolatopsis
rifamycinica (formerly known as Amycolatopsis mediterranei and Streptomyces
mediterranei). It is an antibiotic used to treat infections, including tuberculosis (also
known as TB). It can also be used to prevent infections in those who have been in
contact with serious infections. Rifampicin has 2 pKa since it is a Zwitterion, pKa
1.7 related to 4-hydroxy and pKa 7.9 related to 3-piperazine nitrogen.

Figure 2.1: Chemical structure of rifampicin.

Penicillins can be divided into two groups, namely, (Albarellos et al., 2004)
the natural penicillins, including benzyl penicillin (penicillin G) and its salts
(sodium, potassium, benzathine and procaine), and penicillin V and (Albarellos et
al., 2005) the semi-synthetic penicillins. This second group can be further divided
into three sub-groups: (a) staphylococcal beta-lactamase-resistant penicillins or
isoxazolilpenicillins (oxacillin, cloxacillin and dicloxacillin); (b) broad spectrum or
aminobenzyl penicillins (ampicillin and amoxicillin), and (c) anti-pseudomonal
penicillins (carbenicillin, ticarcillin, piperacillin).

Penicillin was the first microbial metabolite to distinguish between toxicity to

the bacterial cell and toxicity to the mammalian host to permit its use in the systemic
treatment of infections caused by gram-positive and -negative organisms in humans
and animals. The basic structure of penicillin nucleus includes a -lactam ring fused
through nitrogen and adjacent tetrahedral carbon to a second heterocycle, which in
natural penicillin is a five-membered thiazolidine ring that shown in figure 2.2.
Semi-synthetic penicillins are produced starting from 6-aminopenicillanic acid,
which are obtained from culture of Penicillium chrysogenum.

These molecule are more resistant to -lactamase e.g. ampicillin, oxacillin

etc.Penicillin and other -lactams (cephalosporins) inhibit the synthesis of essential
structural components of bacterial cell wall i.e. peptidoglycan which are absent in
mammalian cells. Thus host cell metabolism remains unaffected and penicillins are
regarded as one of the safest and most effective class of antibiotics being used for

bacterial infections. The analysis of degradation products in commercial penicillins

has two-fold importance; firstly in pharmacokinetic studies it is desirable to
distinguish between the drug and any degradation products, secondly allergic
reactions attributed to penicillin may frequently because by such compounds.
Accordingly it is essential to be able to detect the presence of these compounds in the
pharmaceutical compounds.

Figure 2.2: Chemical structure of some important penicillins.

Cephalosporins are -lactam antibiotics, with the same fundamental

structural requirements as penicillin that shown in figure 2.3. Heterocyclic ring fused
to -lactam ring is six membered (dihydrothiazine) in cephalosporins. The fused
rings in -lactams are not coplanar but folded along the C-N bond common to both
rings; less markedly in cephalosporins than in penicillins. The first generation of
cephalosporins are administered parenterally (cephalothin, cefazolin) or orally
(cephalexin, cefadroxil). The second generation includes cefoxitin, cefotetan,
cefamandole and cefuroxime. The third generation includes cefotaxime, ceftazidime,
ceftizoxime, ceftriaxone and ceftiofur. Cefotaxime, ceftazidime, ceftizoxime and
ceftriaxone consistently reach effective antibacterial concentrations in the central
nervous system (CNS) in humans.(Albarellos et al., 2007).

Cephalosporins are used for the treatment of infections caused by most grampositive and -negative bacteria, especially Escherichia coli, Proteus mirabilis and
klebsiella. As discussed in penicillin only cephalosporin C is found in nature isolated

from cultures of fungi other i.e. semi-synthetic cephalosporins are derived from 7aminocephalosporanic acid, product obtained from cephalosporin C hydrolysis.
Literature suggests use of C18 column for chromatographic analysis of this class of
antibiotics. Carbanepem, a newly synthesized -lactam antibiotic, was analysed for
its degradation products by multistage liquid chromatography-electrospray mass
spectrometry.

Figure 2.3: Chemical structure of cephalosporins.

2.1.1 Major principle and definition

While our scientific knowledge of antibiotics has only recently been

developed, the practical application of antibiotics has existed for centuries. The first
known use was by the Chinese about 2,500 years ago. During this time, they
discovered that applying the moldy curd of soybeans to infections had certain
therapeutic benefits. It was so effective that it became a standard treatment. Evidence